The invention relates enhanced Agrobacterium-mediated transformation frequencies of plants due to addition of histones to the plant to be transformed. Methods specific for enhancing monocot transformation frequencies are also disclosed wherein both histones and L-cysteine are factors.
Agrobacterium tumefaciens is a gram negative soil bacterium that has been exploited by plant biologists to introduce foreign DNA into plants. The routine, efficient Agrobacterium-mediated transformation of dicotyledonous plants was first reported in the mid 1980's. Because monocotyledonous plants are not natural hosts for Agrobacterium tumefaciens, the development of transformation systems using this vector for monocots lagged that of dicots. Direct DNA delivery techniques including electroporation, microprojectile bombardment, and silicon carbide fiber treatment were developed for monocot transformation as alternatives to Agrobacterium-based DNA delivery. Production of fertile, transgenic maize plants was first reported in 1990 using microprojectile bombardment. Reports of fertile transgenic maize plant production using electroporation and silicon carbide fiber treatment followed a few years later.
The first well-documented report of fertile transgenic maize plants via Agrobacterium was published by Ishida et al. in 1996, followed by a second report from Negrotto et al. in 2000. Although high frequency Agrobacterium-mediated transformation was reported in those studies, and also in a few maize transformation labs in a private industry, those frequencies have not been reproduced in public maize transformation laboratories. Factors contributing to the lack of reproducibility in the public sector could include: 1) omission of critical details in protocol and media descriptions in published reports, 2) lack of access to specialized binary vectors by public researchers, and 3) reluctance or inability to transfer proprietary information from private industry to the public sector.
The significant advantages of using an Agrobacterium-based transformation system for maize (high frequency transformation, low copy, simple transgene insertion, increased stability of transgene expression, low cost relative to biolistics, and potential to introduce large DNA fragments into the plant genome) make it imperative that optimized protocols be developed, published, and made readily available to maize researchers in the public sector. Although known for this practical application, the actual mechanism of DNA transfer from bacteria to plants is not completely understood. Moreover, there are some limitations on the use of this transforming vector, e.g. difficulties in transforming monocots, and transforming frequencies may be too low to be useful. At present, even some dicots, for example, many Arabidopsis ecotypes and mutants also cannot be easily or efficiently transformed by a root transformation method, generally using Agrobacterium. 
It is believed that Agrobacterium tumefaciens genetically transforms plant cells by transferring a portion of the bacterial Ti-plasmid, designated the T-DNA, to the plant, and integrating the T-DNA into the plant genome. Little is known about the T-DNA integration process, and no plant genes involved in integration have previously been identified. The DNA that is transferred from Agrobacterium to the plant cell is a segment of the Ti, or tumor inducing, plasmid called the T-DNA (transferred DNA). Virulence (vir) genes responsible for T-DNA processing and transfer are reported to lie elsewhere on the Ti plasmid. The role of vir genes in T-DNA processing, the formation of bacterial channels for export of T-DNA, and the attachment of bacteria to the plant cell are reported. In contrast, little is known about the role of plant factors in T-DNA transfer and integration.
Transformations can be transient or stable. Stable transformation is preferred because it is required to produce transgenic plants.
Many plant species are recalcitrant to stable Agrobacterium transformation. These species are, however, easily transiently transformed to express GUS activity or symptoms of viral infection following agroinoculation. Maize BMS cells are readily transiently transformed and could express and process a gus-A-intron transcript encoded by the binary vector pBISN1. Published results implied that, at least in this transformation system, T-DNA could target to maize nuclei and become converted to a double-stranded transcription-competent form. However, the lack of detectable stable transcription suggested that T-DNA integration may be deficient. Thus, making T-DNA integration more efficient and stabilizing T-DNA gene expression are important factors to improve maize transformation.
Integration of exogenous DNA is reported to be improved by delivering the DNA into plant cells with one or more Agrobacterium genes that can encode for proteins within the plant cells. This technique, referred to as “agrolistic transformation” is just an improvement over biolistic transformation by which DNA is delivered to the plants by a non-biological method such as a “gene gun” (biolistic transformation). In this improvement, genes encoding virulence proteins that normally function in Agrobacterium are transferred to the plants along with a T-DNA substrate. The substrate is then acted upon in the plant cell to make a T-DNA molecule. However, the technique described does not include the use of plant genes, or of other factors related herein. The technique was not shown to make a plant more susceptible to transformation. A goal of this method was to increase predictability of the location of integration, not its frequency. Moreover, “agrolistic transformation” is an expensive procedure requiring much infrastructure and resources; one of skill has to go through the laborious process every time to develop a transgenic plant.
The isolation of a putative plant factor has recently been reported. Ballas and Citovsky showed that a plant karyopherin α(AtKAP α) can interact with VirD2 nuclear localization sequences in a yeast two-hybrid interaction system, and is presumably involved in nuclear translocation of the T-complex. Using a similar approach, a tomato type 2C protein phosphatase, DIG3, that can interact with the VirD2 NLS was identified. Unlike AtKAP α, DIG3 plays a negative role in nuclear import. After the T-DNA/T-complex enters the nucleus, it must integrate into the plant chromosome. Plant chromosomal DNA is packaged into nucleosomes consisting primarily of histone proteins. The incoming T-DNA may have to interact with this nucleosome structure during the integration process. However, T-DNA may preferentially integrate into transcribed regions of the genome. These regions are believed to be temporarily free of histones. How exactly T-DNA integration takes place is unknown. Recent reports have implicated involvement of VirD2 protein in the T-DNA integration process.
Several ecotypes of the dicot Arabidopsis are resistant to Agrobacterium transformation. Transforming the transformation resistant rat5 mutant of Arabidopsis with a wild-type RAT5 (histone H2A) gene was reported by the inventors to complement the mutant phenotype.
In monocots, maize is the most studied model plant that has important economic value. Although genetic transformation systems for the maize have been established in private laboratories, the lack of such systems is still a key limitation for public researchers. This is because most public research groups do not have access to the resources and infrastructure necessary for maize transformation by currently available procedures. In addition, the current technology has serious limitations, including low efficiency and throughput, difficulty with inbred line transformation, unpredictable transgene copy numbers and integrity, and undesirable transgene silencing during development and over generations.
Because fertile transgenic maize (Zea mays) was first produced using the biolistic gun, maize transformation technology has served as an important tool in germplasm development and research addressing fundamental biological questions through the study of transgenic maize. Recent reports have demonstrated that Agrobacterium tumefaciens-mediated maize transformation may offer a better alternative than the biolistic gun for delivery of transgenes to maize. This gene delivery system results in a greater proportion of stable, low-copy number transgenic events than does the biolistic gun, offers the possibility of transferring larger DNA segments into recipient cells, and is highly efficient. Reproducible protocols for A. tumefaciens-mediated maize transformation have used super binary vectors, in which the A. tumefaciens strain carries extra copies of virB, virC, and virG, to infect immature zygotic embryos of the inbred line A188 or the hybrid line Hi II. Hi II immature zygotic embryos were transformed by the inventor at an average efficiency of 5.8% using the A. tumefaciens super binary vector in strain LBA4404. Because the cost of licensing this proprietary technology for use on a broader scale may be prohibitive to a public sector laboratory, the inventors implemented an A. tumefaciens standard binary (non-super binary) vector system to transform maize Hi II immature zygotic embryos. Stable transformation of maize using a standard binary vector to infect shoot meristems was reported previously, but adoption of this method was hindered by its lack of robustness. Development of a reproducible and efficient method for transforming maize using a standard binary vector will not only provide researchers with the benefits already outlined, it would also facilitate vector construction when compared with the super binary vector. Final assembly of a super binary vector system involves co-integration of the gene of interest into a large plasmid (pSB1) in A. tumefaciens strain LBA4404 via homologous recombination. In contrast, assembly of a standard binary vector does not require this additional step, making it a more efficient way to confirm the introduction of a gene of interest into an A. tumefaciens strain.
Transformation of maize (Zea mays) using an Agrobacterium tumefaciens standard binary (non-super binary) vector system was achieved by the inventors. Immature zygotic embryos of the hybrid line Hi II were infected with A. tumefaciens strain EHA101 harboring a standard binary vector and cocultivated in the presence of 400 mg/L L-cysteine. Inclusion of L-cysteine in cocultivation medium led an improvement in transient-glucuronidase expression observed in targeted cells and a significant increase in stable transformation efficiency, but was associated with a decrease in embryo response after cocultivation. The average stable transformation efficiency (no. of bialaphos-resistant events recovered per 100 embryos infected) was 5.5%. Southern-blot and progeny analyses confirmed the integration, expression, and inheritance of the bar and gus transgenes in R0, R1, and R2 generations of transgenic events. Fertile, stable transgenic maize was routinely produced using an A. tumefaciens standard binary vector system.
The level of stable transformation achieved is attributed to supplementation of cocultivation medium with 400 mg/L Cys. This antioxidant treatment also increased T-DNA delivery to embryogenic-competent scutellum cells of infected embryos. A similar increase in transient gus gene expression, followed by an increase in stable transformation efficiency, was reported in soybean cotyledonary node explants infected with A. tumefaciens and cocultivated on medium supplemented with Cys.
Contrary to expectations, the increase in stable transformation efficiency observed with the 400 mg/L Cys treatment was associated with a decrease in the proportion of embryos giving rise to embryogenic callus compared with the 0 mg/L Cys treatment. This reduction in embryo response is not related to the plant-pathogen interaction per se because noninfected embryos also exhibited reduced response on 400 mg/L Cys. It is likely that Cys concentrations as high as 400 mg/L are toxic to maize cells. A similar negative impact of 80 mg/L Cys on embryogenesis in Japonica rice explants was reported by Enriquez-Obregon et al. (1999). Comparable stable transformation rates were achieved using Cys concentrations as low as 100 mg/L, and this treatment was associated with better embryo recovery after cocultivation than that observed using the 400 mg/L Cys treatment.
A. tumefaciens-mediated maize transformation using a standard binary vector system is reproducible although variability in experimental efficiency persists. Using cocultivation medium within 7 d of preparation minimizes this variability. Average transformation efficiency is about 5.5%.
Information on plant factors and other factors affecting Agrobacterium transformation frequencies in plants is needed to improve performance of this method in both dicots and monocots.